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1.
Adv Healthc Mater ; : e2402024, 2024 Sep 03.
Article in English | MEDLINE | ID: mdl-39226530

ABSTRACT

In the pursuit of new wound care products, researchers are exploring methods to improve wound healing through exogenous wound healing products. However, diverging from this conventional approach, this work has developed an endogenous support system for wound healing, drawing inspiration from the body's innate healing mechanisms governed by the sequential release of metal ions by body at wound site to promote different stages of wound healing. This work engineers a multi-ion-releasing sprayable hydrogel system, to mimic this intricate process, representing the next evolutionary step in wound care products. It comprises Alginate (Alg) and Fibrin (Fib) hydrogel infused with Polylactic acid (PLA) polymeric microcarriers encapsulating multi (calcium, copper, and zinc) nanoparticles (Alg-Fib-PLA-nCMB). Developed sprayable Alg-Fib-PLA-nCMB hydrogel show sustained release of beneficial multi metallic ions at wound site, offering a range of advantages including enhanced cellular function, antibacterial properties, and promotion of crucial wound healing processes like cell migration, ROS mitigation, macrophage polarization, collagen deposition, and vascular regeneration. In a comparative study with a commercial product (Midstress spray), developed Alg-Fib-PLA-nCMB hydrogel demonstrates superior wound healing outcomes in a rat model, indicating its potential for next generation wound care product, addressing critical challenges and offering a promising avenue for future advancements in the wound management.

2.
J Chem Phys ; 161(9)2024 Sep 07.
Article in English | MEDLINE | ID: mdl-39225536

ABSTRACT

Biomolecular condensates are viscoelastic materials defined by time-dependent, sequence-specific complex shear moduli. Here, we show that viscoelastic moduli can be computed directly using a generalization of the Rouse model that leverages information regarding intra- and inter-chain contacts, which we extract from equilibrium configurations of lattice-based Metropolis Monte Carlo (MMC) simulations of phase separation. The key ingredient of the generalized Rouse model is a graph Laplacian that we compute from equilibrium MMC simulations. We compute two flavors of graph Laplacians, one based on a single-chain graph that accounts only for intra-chain contacts, and the other referred to as a collective graph that accounts for inter-chain interactions. Calculations based on the single-chain graph systematically overestimate the storage and loss moduli, whereas calculations based on the collective graph reproduce the measured moduli with greater fidelity. However, in the long time, low-frequency domain, a mixture of the two graphs proves to be most accurate. In line with the theory of Rouse and contrary to recent assertions, we find that a continuous distribution of relaxation times exists in condensates. The single crossover frequency between dominantly elastic vs dominantly viscous behaviors does not imply a single relaxation time. Instead, it is influenced by the totality of the relaxation modes. Hence, our analysis affirms that viscoelastic fluid-like condensates are best described as generalized Maxwell fluids. Finally, we show that the complex shear moduli can be used to solve an inverse problem to obtain the relaxation time spectra that underlie the dynamics within condensates. This is of practical importance given advancements in passive and active microrheology measurements of condensate viscoelasticity.


Subject(s)
Biomolecular Condensates , Monte Carlo Method , Viscosity , Biomolecular Condensates/chemistry , Elasticity
3.
Nat Commun ; 15(1): 7686, 2024 Sep 03.
Article in English | MEDLINE | ID: mdl-39227569

ABSTRACT

The internal microenvironment of a living cell is heterogeneous and comprises a multitude of organelles with distinct biochemistry. Amongst them are biomolecular condensates, which are membrane-less, phase-separated compartments enriched in system-specific proteins and nucleic acids. The heterogeneity of the cell engenders the presence of multiple spatiotemporal gradients in chemistry, charge, concentration, temperature, and pressure. Such thermodynamic gradients can lead to non-equilibrium driving forces for the formation and transport of biomolecular condensates. Here, we report how ion gradients impact the transport processes of biomolecular condensates on the mesoscale and biomolecules on the microscale. Utilizing a microfluidic platform, we demonstrate that the presence of ion concentration gradients can accelerate the transport of biomolecules, including nucleic acids and proteins, via diffusiophoresis. This hydrodynamic transport process allows localized enrichment of biomolecules, thereby promoting the location-specific formation of biomolecular condensates via phase separation. The ion gradients further impart directional motility of condensates, allowing them to exhibit enhanced diffusion along the gradient. Coupled with a reentrant phase behavior, the gradient-induced enhanced motility leads to a dynamical redistribution of condensates that ultimately extends their lifetime. Together, our results demonstrate diffusiophoresis as a non-equilibrium thermodynamic force that governs the formation and transport of biomolecular condensates.


Subject(s)
Biomolecular Condensates , Biomolecular Condensates/metabolism , Biomolecular Condensates/chemistry , Diffusion , Hydrodynamics , Nucleic Acids/metabolism , Nucleic Acids/chemistry , Thermodynamics , Proteins/metabolism , Proteins/chemistry , Biological Transport , Microfluidics/methods , Phase Separation
4.
J Control Release ; 374: 349-368, 2024 Aug 22.
Article in English | MEDLINE | ID: mdl-39111600

ABSTRACT

Extracellular vesicles (EVs), or exosomes, play important roles in physiological and pathological cellular communication and have gained substantial traction as biological drug carriers. EVs contain both short and long non-coding RNAs that regulate gene expression and epigenetic processes. To fully capitalize on the potential of EVs as drug carriers, it is important to study and understand the intricacies of EV function and EV RNA-based communication. Here we developed a genetically encodable RNA-based biomaterial, termed EXO-Probe, for tracking EV RNAs. The EXO-Probe comprises an EV-loading RNA sequence (EXO-Code), fused to a fluorogenic RNA Mango aptamer for RNA imaging. This fusion construct allowed the visualization and tracking of EV RNA and colocalization with markers of multivesicular bodies; imaging RNA within EVs, and non-destructive quantification of EVs. Overall, the new RNA-based biomaterial provides a useful and versatile means to interrogate the role of EVs in cellular communication via RNA trafficking to EVs and to study cellular sorting decisions. The system will also help lay the foundation to further improve the therapeutic efficacy of EVs as drug carriers.

5.
Res Sq ; 2024 Jul 19.
Article in English | MEDLINE | ID: mdl-39070659

ABSTRACT

Intracellular aggregation of repeat expanded RNA has been implicated in many neurological disorders. Here, we study the role of biomolecular condensates on irreversible RNA clustering. We find that physiologically relevant and disease-associated repeat RNAs spontaneously undergo an age-dependent percolation transition inside multi-component protein-nucleic acid condensates to form nanoscale clusters. Homotypic RNA clusters drive the emergence of multiphasic condensate structures with an RNA-rich solid core surrounded by an RNA-depleted fluid shell. The timescale of the RNA clustering, which drives a liquid-to-solid transition of biomolecular condensates, is determined by the sequence features, stability of RNA secondary structure, and repeat length. Importantly, G3BP1, the core scaffold of stress granules, introduces heterotypic buffering to homotypic RNA-RNA interactions and impedes intra-condensate RNA clustering in an ATP-independent manner. Our work suggests that biomolecular condensates can act as sites for RNA aggregation. It also highlights the functional role of RNA-binding proteins in suppressing aberrant RNA phase transitions.

6.
bioRxiv ; 2024 Jun 13.
Article in English | MEDLINE | ID: mdl-38915484

ABSTRACT

In vitro facsimiles of biomolecular condensates are formed by different types of intrinsically disordered proteins including prion-like low complexity domains (PLCDs). PLCD condensates are viscoelastic materials defined by time-dependent, sequence-specific complex shear moduli. Here, we show that viscoelastic moduli can be computed directly using a generalization of the Rouse model and information regarding intra- and inter-chain contacts that is extracted from equilibrium configurations of lattice-based Metropolis Monte Carlo (MMC) simulations. The key ingredient of the generalized Rouse model is the Zimm matrix that we compute from equilibrium MMC simulations. We compute two flavors of Zimm matrices, one referred to as the single-chain model that accounts only for intra-chain contacts, and the other referred to as a collective model, that accounts for inter-chain interactions. The single-chain model systematically overestimates the storage and loss moduli, whereas the collective model reproduces the measured moduli with greater fidelity. However, in the long time, low-frequency domain, a mixture of the two models proves to be most accurate. In line with the theory of Rouse, we find that a continuous distribution of relaxation times exists in condensates. The single crossover frequency between dominantly elastic versus dominantly viscous behaviors is influenced by the totality of the relaxation modes. Hence, our analysis suggests that viscoelastic fluid-like condensates are best described as generalized Maxwell fluids. Finally, we show that the complex shear moduli can be used to solve an inverse problem to obtain distributions of relaxation times that underlie the dynamics within condensates.

7.
bioRxiv ; 2024 Jun 13.
Article in English | MEDLINE | ID: mdl-38915678

ABSTRACT

Intracellular aggregation of repeat expanded RNA has been implicated in many neurological disorders. Here, we study the role of biomolecular condensates on irreversible RNA clustering. We find that physiologically relevant and disease-associated repeat RNAs spontaneously undergo an age-dependent percolation transition inside multi-component protein-nucleic acid condensates to form nanoscale clusters. Homotypic RNA clusters drive the emergence of multiphasic condensate structures with an RNA-rich solid core surrounded by an RNA-depleted fluid shell. The timescale of the RNA clustering, which drives a liquid-to-solid transition of biomolecular condensates, is determined by the sequence features, stability of RNA secondary structure, and repeat length. Importantly, G3BP1, the core scaffold of stress granules, introduces heterotypic buffering to homotypic RNA-RNA interactions and impedes intra-condensate RNA clustering in an ATP-independent manner. Our work suggests that biomolecular condensates can act as sites for RNA aggregation. It also highlights the functional role of RNA-binding proteins in suppressing aberrant RNA phase transitions.

8.
Sci Adv ; 10(7): eadi6539, 2024 Feb 16.
Article in English | MEDLINE | ID: mdl-38363841

ABSTRACT

The form and function of biomolecular condensates are intimately linked to their material properties. Here, we integrate microrheology with molecular simulations to dissect the physical determinants of condensate fluid phase dynamics. By quantifying the timescales and energetics of network relaxation in a series of heterotypic viscoelastic condensates, we uncover distinctive roles of sticker motifs, binding energy, and chain length in dictating condensate dynamical properties. We find that the mechanical relaxation times of condensate-spanning networks are determined by both intermolecular interactions and chain length. We demonstrate, however, that the energy barrier for network reconfiguration, termed flow activation energy, is independent of chain length and only varies with the strengths of intermolecular interactions. Biomolecular diffusion in the dense phase depends on a complex interplay between viscoelasticity and flow activation energy. Our results illuminate distinctive roles of chain length and sequence-specific multivalent interactions underlying the complex material and transport properties of biomolecular condensates.


Subject(s)
Biomolecular Condensates , Hydrodynamics , Physical Phenomena , Diffusion , Physical Examination
9.
Nat Commun ; 15(1): 1168, 2024 Feb 07.
Article in English | MEDLINE | ID: mdl-38326345

ABSTRACT

Prion-like domains (PLDs) are low-complexity protein sequences enriched within nucleic acid-binding proteins including those involved in transcription and RNA processing. PLDs of FUS and EWSR1 play key roles in recruiting chromatin remodeler mammalian SWI/SNF (mSWI/SNF) complex to oncogenic FET fusion protein condensates. Here, we show that disordered low-complexity domains of multiple SWI/SNF subunits are prion-like with a strong propensity to undergo intracellular phase separation. These PLDs engage in sequence-specific heterotypic interactions with the PLD of FUS in the dilute phase at sub-saturation conditions, leading to the formation of PLD co-condensates. In the dense phase, homotypic and heterotypic PLD interactions are highly cooperative, resulting in the co-mixing of individual PLD phases and forming spatially homogeneous condensates. Heterotypic PLD-mediated positive cooperativity in protein-protein interaction networks is likely to play key roles in the co-phase separation of mSWI/SNF complex with transcription factors containing homologous low-complexity domains.


Subject(s)
Prions , Animals , Prions/metabolism , Transcription Factors/metabolism , Chromatin , Mammals/genetics , Chromatin Assembly and Disassembly
10.
bioRxiv ; 2024 Feb 17.
Article in English | MEDLINE | ID: mdl-37461689

ABSTRACT

The internal microenvironment of a living cell is heterogeneous and comprises a multitude of organelles with distinct biochemistry. Amongst them are biomolecular condensates, which are membrane-less, phase-separated compartments enriched in system-specific proteins and nucleic acids. The heterogeneity of the cell engenders the presence of multiple spatiotemporal gradients in chemistry, charge, concentration, temperature, and pressure. Such thermodynamic gradients can lead to non-equilibrium driving forces for the formation and transport of biomolecular condensates. Here, we report how ion gradients impact the transport processes of biomolecular condensates on the mesoscale and biomolecules on the microscale. Utilizing a microfluidic platform, we demonstrate that the presence of ion concentration gradients can accelerate the transport of biomolecules, including nucleic acids and proteins, via diffusiophoresis. This hydrodynamic transport process allows localized enrichment of biomolecules, thereby promoting the location-specific formation of biomolecular condensates via phase separation. The ion gradients further impart active motility of condensates, allowing them to exhibit enhanced diffusion along the gradient. Coupled with a reentrant phase behavior, the gradient-induced active motility leads to a dynamical redistribution of condensates that ultimately extends their lifetime. Together, our results demonstrate diffusiophoresis as a non-equilibrium thermodynamic force that governs the formation and transport of biomolecular condensates.

11.
Trends Biochem Sci ; 49(2): 101-104, 2024 02.
Article in English | MEDLINE | ID: mdl-37949765

ABSTRACT

Intrinsically disordered regions (IDRs) within human proteins play critical roles in cellular information processing, including signaling, transcription, stress response, DNA repair, genome organization, and RNA processing. Here, we summarize current challenges in the field and propose cutting-edge approaches to address them in physiology and disease processes, with a focus on cancer.


Subject(s)
Intrinsically Disordered Proteins , Humans , Intrinsically Disordered Proteins/metabolism , Biophysics , Biology
12.
Nat Chem ; 15(12): 1693-1704, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37932412

ABSTRACT

Co-phase separation of RNAs and RNA-binding proteins drives the biogenesis of ribonucleoprotein granules. RNAs can also undergo phase transitions in the absence of proteins. However, the physicochemical driving forces of protein-free, RNA-driven phase transitions remain unclear. Here we report that various types of RNA undergo phase separation with system-specific lower critical solution temperatures. This entropically driven phase separation is an intrinsic feature of the phosphate backbone that requires Mg2+ ions and is modulated by RNA bases. RNA-only condensates can additionally undergo enthalpically favourable percolation transitions within dense phases. This is enabled by a combination of Mg2+-dependent bridging interactions between phosphate groups and RNA-specific base stacking and base pairing. Phase separation coupled to percolation can cause dynamic arrest of RNAs within condensates and suppress the catalytic activity of an RNase P ribozyme. Our work highlights the need to incorporate RNA-driven phase transitions into models for ribonucleoprotein granule biogenesis.


Subject(s)
RNA, Catalytic , RNA , Temperature , RNA-Binding Proteins , Phosphates , Phase Transition
13.
Res Sq ; 2023 Jul 27.
Article in English | MEDLINE | ID: mdl-37546778

ABSTRACT

The internal microenvironment of a living cell is heterogeneous and comprises a multitude of organelles with distinct biochemistry. Amongst them are biomolecular condensates, which are membrane-less, phase-separated compartments enriched in system-specific proteins and nucleic acids. The heterogeneity of the cell engenders the presence of multiple spatiotemporal gradients in chemistry, charge, concentration, temperature, and pressure. Such thermodynamic gradients can lead to non-equilibrium driving forces for the formation and transport of biomolecular condensates. Here, we report how ion gradients impact the transport processes of biomolecular condensates on the mesoscale and biomolecules on the microscale. Utilizing a microfluidic platform, we demonstrate that the presence of ion concentration gradients can accelerate the transport of biomolecules, including nucleic acids and proteins, via diffusiophoresis. This hydrodynamic transport process allows localized enrichment of biomolecules, thereby promoting the location-specific formation of biomolecular condensates via phase separation. The ion gradients further impart active motility of condensates, allowing them to exhibit enhanced diffusion along the gradient. Coupled with reentrant phase behavior, the gradient-induced active motility leads to a dynamical redistribution of condensates that ultimately extends their lifetime. Together, our results demonstrate diffusiophoresis as a non-equilibrium thermodynamic force that governs the formation and active transport of biomolecular condensates.

14.
bioRxiv ; 2023 Nov 22.
Article in English | MEDLINE | ID: mdl-37090622

ABSTRACT

Prion-like domains (PLDs) are low-complexity protein sequences enriched within nucleic acid-binding proteins including those involved in transcription and RNA processing. PLDs of FUS and EWSR1 play key roles in recruiting chromatin remodeler mammalian SWI/SNF complex to oncogenic FET fusion protein condensates. Here, we show that disordered low-complexity domains of multiple SWI/SNF subunits are prion-like with a strong propensity to undergo intracellular phase separation. These PLDs engage in sequence-specific heterotypic interactions with the PLD of FUS in the dilute phase at sub-saturation conditions, leading to the formation of PLD co-condensates. In the dense phase, homotypic and heterotypic PLD interactions are highly cooperative, resulting in the co-mixing of individual PLD phases and forming spatially homogeneous co-condensates. Heterotypic PLD-mediated positive cooperativity in protein-protein interaction networks is likely to play key roles in the co-phase separation of mSWI/SNF complex with transcription factors containing homologous low-complexity domains.

15.
bioRxiv ; 2023 Dec 26.
Article in English | MEDLINE | ID: mdl-37066350

ABSTRACT

Biomolecular condensates are viscoelastic materials. Here, we report results from investigations into molecular-scale determinants of sequence-encoded and age-dependent viscoelasticity of condensates formed by prion-like low-complexity domains (PLCDs). The terminally viscous forms of PLCD condensates are Maxwell fluids. Measured viscoelastic moduli of these condensates are reproducible using a Rouse-Zimm model that accounts for the network-like organization engendered by reversible physical crosslinks among PLCDs in the dense phase. Measurements and computations show that the strengths of aromatic inter-sticker interactions determine the sequence-specific amplitudes of elastic and viscous moduli as well as the timescales over which elastic properties dominate. PLCD condensates also undergo physical aging on sequence-specific timescales. This is driven by mutations to spacer residues that weaken the metastability of terminally viscous phases. The aging of PLCD condensates is accompanied by disorder-to-order transitions, leading to the formation of non-fibrillar, beta-sheet-containing, semi-crystalline, terminally elastic, Kelvin-Voigt solids. Our results suggest that sequence grammars, which refer to the identities of stickers versus spacers in PLCDs, have evolved to afford control over the metastabilities of terminally viscous fluid phases of condensates. This selection can, in some cases, render barriers for conversion from metastable fluids to globally stable solids to be insurmountable on functionally relevant timescales.

16.
Am J Forensic Med Pathol ; 44(2): 136-139, 2023 Jun 01.
Article in English | MEDLINE | ID: mdl-36989081

ABSTRACT

ABSTRACT: Suicide rapidly increased in the United States by 30% from 2000 to 2020, accounting for more than 800,000 deaths ( Neurosci Res Program Bull . 1972; 10: 384-8). Studies have shown that there are a multitude of underlying issues, including mental illness, that elevate an individual's risk of dying by suicide ( CDC WONDER: Underlying cause of death, 1999-2019 . Atlanta, GA: US Department of Health and Human Services, CDC; 2020). Presented here is a case of Bing Neel syndrome (BNS) found in a 69-year-old man who died by suicide by jumping off a 135' bridge. His medical history was significant for traumatic brain injury, Waldenstrom macroglobulinemia (WM), major depressive disorder, suicidal ideation, and anxiety. Bing Neel syndrome is a rare central nervous system complication of WM. His wife reported an abrupt mental deterioration starting 5 years before his death, characterized by paranoia, depression, and insomnia. He had been a high-functioning university professor. His decline culminated with the loss of independence in his activities of daily living. At autopsy, it was found that he experienced blunt force injuries related to the fall, causing his death. A neuropathologic examination revealed a brisk and fulminant clonal CD20 + /immunoglobulin M+ lymphocytic infiltrate, involving all sampled regions of his brain, consistent with WM. This workup was critical to obtaining an accurate pathologic diagnosis of BNS and understanding his full clinical status before death. Although BNS was not the proximate cause of death, this diagnosis aided the death investigation as a causal factor in his suicidality and was vital to providing his family closure.


Subject(s)
Depressive Disorder, Major , Psychotic Disorders , Suicide , Waldenstrom Macroglobulinemia , Humans , Male , Animals , Cattle , Aged , Suicidal Ideation , Activities of Daily Living , Waldenstrom Macroglobulinemia/complications , Waldenstrom Macroglobulinemia/diagnosis , Waldenstrom Macroglobulinemia/pathology , Psychotic Disorders/complications
17.
Methods Mol Biol ; 2563: 199-213, 2023.
Article in English | MEDLINE | ID: mdl-36227474

ABSTRACT

Liquid-liquid phase separation of protein and RNA complexes into biomolecular condensates has emerged as a ubiquitous phenomenon in living systems. These protein-RNA condensates are thought to be involved in many biological functions in all forms of life. One of the most sought-after properties of these condensates is their dynamical properties, as they are a major determinant of condensate physiological function and disease processes. Measurement of the diffusion dynamics of individual components in a multicomponent biomolecular condensate is therefore routinely performed. Here, we outline the experimental procedure for performing in-droplet fluorescence correlation spectroscopy (FCS) measurements to extract the diffusion coefficient of individual molecules within a biomolecular condensate in vitro. Unlike more common experiments such as fluorescence recovery after photobleaching (FRAP), where data interpretation is not straightforward and strictly model dependent, FCS offers a robust and more accurate way to quantify biomolecular diffusion rates in the dense phase. The small observation volume allows FCS experiments to report on the local diffusion coefficient within a spatial resolution of <1 µm, making it ideal for probing spatial inhomogeneities within condensates as well as variable dynamics within subcompartments of multiphasic condensates.


Subject(s)
Nucleic Acids , Biomolecular Condensates , Fluorescence Recovery After Photobleaching , RNA , Spectrum Analysis
18.
Trends Cell Biol ; 32(8): 681-695, 2022 08.
Article in English | MEDLINE | ID: mdl-35484036

ABSTRACT

Biomolecular condensates are membraneless organelles (MLOs) that are enriched in specific proteins and nucleic acids, compartmentalized to perform biochemical functions. Such condensates are formed by phase separation (PS) enabled by protein domains that allow multivalent interactions. Chromosomal translocation-derived in-frame gene fusions often generate proteins with non-native domain combinations that rewire protein-protein interaction networks. Several recent studies have shown that, for a subset of these fusion proteins, pathogenesis can be driven by the ability of the fusion protein to undergo phase transitions at non-physiological cellular locations to form ectopic condensates. We highlight how such ectopic phase transitions can alter biological processes and posit that dysfunction via protein PS at non-physiological locations represents a generic route to oncogenic transformation.


Subject(s)
Neoplasms , Nucleic Acids , Humans , Neoplasms/genetics , Neoplasms/metabolism , Nucleic Acids/metabolism , Organelles/metabolism , Phase Transition , Proteins/metabolism
19.
iScience ; 25(4): 104105, 2022 Apr 15.
Article in English | MEDLINE | ID: mdl-35378855

ABSTRACT

Proteomic studies have shown that cellular condensates are frequently enriched in diverse RNA molecules, which is suggestive of mechanistic links between phase separation and transcriptional activities. Here, we report a systematic experimental and computational study of thermodynamic landscapes and interfacial properties of protein-RNA condensates. We have studied the affinity of protein-RNA condensation as a function of variable RNA sequence length and RNA-protein stoichiometry under different ionic environments and external crowding. We have chosen the PolyU sequences for RNA and arginine/glycine-rich intrinsically disordered peptide (RGG) for proteins as a model system of RNA-protein condensates, which we then investigate through in vitro microscopy measurements and coarse-grained molecular dynamics simulations. We find that crowding and RNA chain length can have a major stabilizing effect on the condensation. We also find that the RNA-protein charge ratio is a crucial variable controlling stability, interfacial properties, and the reentrant phase behavior of RGG-RNA mixtures.

20.
Soft Matter ; 18(7): 1342-1349, 2022 Feb 16.
Article in English | MEDLINE | ID: mdl-34984429

ABSTRACT

Liquid-liquid phase separation (LLPS) of multivalent biopolymers is a ubiquitous process in biological systems and is of importance in bio-mimetic soft matter design. The phase behavior of biomolecules, such as proteins and nucleic acids, is typically encoded by the primary chain sequence and regulated by solvent properties. One of the most important physical modulators of LLPS is temperature. Solutions of proteins and/or nucleic acids have been shown to undergo liquid-liquid phase separation either upon cooling (with an upper critical solution temperature, UCST) or upon heating (with a lower critical solution temperature, LCST). However, many theoretical frameworks suggest the possibility of more complex temperature-dependent phase behaviors, such as an hourglass or a closed-loop phase diagram with concurrent UCST and LCST transitions. Here, we report that RNA-polyamine mixtures undergo a reentrant phase separation with temperature. Specifically, at low temperatures, RNA-polyamine mixtures form a homogenous phase. Increasing the temperature leads to the formation of RNA-polyamine condensates. A further increase in temperature leads to the dissolution of condensates, rendering a reentrant homogenous phase. This dual-response phase separation of RNA is not unique to polyamines but also observed with short cationic peptides. The immiscibility gap is controlled by the charge of the polycation, salt concentration, and mixture composition. Based on the existing theories of complex coacervation, our results point to a complex interplay between desolvation entropy, ion-pairing, and electrostatic interactions in dictating the closed-loop phase behavior of RNA-polycation mixtures.


Subject(s)
Proteins , RNA , Phase Transition , Polyelectrolytes , Temperature
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